Knowledge of the kinds and numbers of nuclear point mutations in human tissues is essential to the understanding of the mutation mechanisms underlying genetic diseases. However, nuclear point mutant fractions in normal humans are so low that few methods exist to measure them. We have now developed a means to scan for point mutations in 100 bp nuclear single copy sequences at mutant fractions as low as 10–6. Beginning with about 108 human cells we first enrich for the desired nuclear sequence 10 000-fold from the genomic DNA by sequence-specific hybridization coupled with a biotin–streptavidin capture system. We next enrich for rare mutant sequences 100-fold against the wild-type sequence by wide bore constant denaturant capillary electrophoresis (CDCE). The mutant-enriched sample is subsequently amplified by high fidelity PCR using fluorescein-labeled primers. Amplified mutant sequences are further enriched via two rounds of CDCE coupled with high fidelity PCR. Individual mutants, seen as distinct peaks on CDCE, are then isolated and sequenced. We have tested this approach by measuring N-methyl-N ′-nitro-N-nitrosoguanidine (MNNG)-induced point mutations in a 121 bp sequence of the adenomatous polyposis coli gene (APC) in human lymphoblastoid MT1 cells. Twelve different MNNG-induced GC→AT transitions were reproducibly observed in MNNG-treated cells at mutant fractions between 2 × 10–6 and 9 × 10–6. The sensitivity of this approach was limited by the fidelity of Pfu DNA polymerase, which created 14 different GC→TA transversions at a mutant fraction equivalent to ~10–6 in the original samples. The approach described herein should be general for all DNA sequences suitable for CDCE analysis. Its sensitivity and capacity would permit detection of stem cell mutations in tissue sectors consisting of ~108 cells. 相似文献
In the soybean cultivar Suweon 97, BCMV-resistance gene was fine-mapped to a 58.1-kb region co-localizing with the Soybean mosaic virus (SMV)-resistance gene, Rsv1-h raising a possibility that the same gene is utilized against both viral pathogens.
Abstract
Certain soybean cultivars exhibit resistance against soybean mosaic virus (SMV) or bean common mosaic virus (BCMV). Although several SMV-resistance loci have been reported, the understanding of the mechanism underlying BCMV resistance in soybean is limited. Here, by crossing a resistant cultivar Suweon 97 with a susceptible cultivar Williams 82 and inoculating 220 F2 individuals with a BCMV strain (HZZB011), we observed a 3:1 (resistant/susceptible) segregation ratio, suggesting that Suweon 97 possesses a single dominant resistance gene against BCMV. By performing bulked segregant analysis with 186 polymorphic simple sequence repeat (SSR) markers across the genome, the resistance gene was determined to be linked with marker BARSOYSSR_13_1109. Examining the genotypes of nearby SSR markers on all 220 F2 individuals then narrowed down the gene between markers BARSOYSSR_13_1109 and BARSOYSSR_13_1122. Furthermore, 14 previously established F2:3 lines showing crossovers between the two markers were assayed for their phenotypes upon BCMV inoculation. By developing six more SNP (single nucleotide polymorphism) markers, the resistance gene was finally delimited to a 58.1-kb interval flanked by BARSOYSSR_13_1114 and SNP-49. Five genes were annotated in this interval of the Williams 82 genome, including a characteristic coiled-coil nucleotide-binding site-leucine-rich repeat (CC-NBS-LRR, CNL)-type of resistance gene, Glyma13g184800. Coincidentally, the SMV-resistance allele Rsv1-h was previously mapped to almost the same region, thereby suggesting that soybean Suweon 97 likely relies on the same CNL-type R gene to resist both viral pathogens.
Corynebacterium diphtheriae, the causative agent of diphtheria, is well-investigated in respect to toxin production, while little is known about C. diphtheriae factors crucial for colonization of the host. In this study, we investigated the function of surface-associated protein DIP1281,
previously annotated as hypothetical invasion-associated protein. 相似文献
Benzo[a]pyrene is an important environmental mutagen and carcinogen. Its metabolism in cells yields the mutagenic, key ultimate carcinogen 7R,8S,9S,10R-anti-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide, (+)-anti-BPDE, which reacts via its 10-position with N2-dG in DNA to form the adduct (+)-trans-anti-BPDE-N2-dG. To gain molecular insights into BPDE-induced mutagenesis, we examined in vivo translesion synthesis and mutagenesis in yeast cells of a site-specific 10S (+)-trans-anti-BPDE-N2-dG adduct and the stereoisomeric 10R (−)-trans-anti-BPDE-N2-dG adduct. In wild-type cells, bypass products consisted of 76% C, 14% A and 7% G insertions opposite (+)-trans-anti-BPDE-N2-dG; and 89% C, 4% A and 4% G insertions opposite (−)-trans-anti-BPDE-N2-dG. Translesion synthesis was reduced by ~26–37% in rad30 mutant cells lacking Polη, but more deficient in rev1 and almost totally deficient in rev3 (lacking Polζ) mutants. C insertion opposite the lesion was reduced by ~24–33% in rad30 mutant cells, further reduced in rev1 mutant, and mostly disappeared in the rev3 mutant strain. The insertion of A was largely abolished in cells lacking either Polη, Polζ or Rev1. The insertion of G was not detected in either rev1 or rev3 mutant cells. The rad30 rev3 double mutant exhibited a similar phenotype as the single rev3 mutant with respect to translesion synthesis and mutagenesis. These results show that while the Polζ pathway is generally required for translesion synthesis and mutagenesis of the (+)- and (−)-trans-anti-BPDE-N2-dG DNA adducts, Polη, Polζ and Rev1 together are required for G→T transversion mutations, a major type of mutagenesis induced by these lesions. Based on biochemical and genetic results, we present mechanistic models of translesion synthesis of these two DNA adducts, involving both the one-polymerase one-step and two-polymerase two-step models. 相似文献
Corynebacterium diphtheriae strains lyso-genic for phage β are able to produce diphtheria toxin. This article describes evidence suggesting that the toxin structural gene is part of the phage genome. 相似文献
The aerobic gram positive bacterium Corynebacterium diphtheriae causes diphtheria, a respiratory tract illness characterized by symptoms such as sore throat, low fever, and an adherent membrane on the tonsils, pharynx, and/or nasal cavity. Therefore, it is important to develop preventive vaccines for diphtheria. The availability of the 2,488,635 bp long complete sequence for the C. diphtheriae genome provides an opportunity to understand cell mediated immune response using Computational Biology tools from the bacterial proteome sequence data. We selected 355 membrane proteins from the C. diphtheriae proteome using annotation data to identify potential HLA-DRB1 binding short peptide using modeling, simulations and predictions. This exercise identified 30 short peptides in membrane proteins showing binding capability to HLA-DRB1 alleles. These peptides serve as outline for the understanding of cell mediated immune response to C. diphtheriae. It should be noted that the predicted data to be verified using binding assays for further consideration. 相似文献
The grain color of wheat affects not only the brightness of flour, but also tolerance to preharvest sprouting. Grain color
is controlled by dominant R-1 genes located on the long arm of hexaploid wheat chromosomes 3A, 3B, and 3D (R-A1, R-B1, and R-D1, respectively). The red pigment of the grain coat is composed of catechin and proanthocyanidin (PA), which are synthesized
via the flavonoid biosynthetic pathway. We isolated the Tamyb10-A1, Tamyb10-B1, and Tamyb10-D1 genes, located on chromosomes 3A, 3B, and 3D, respectively. These genes encode R2R3-type MYB domain proteins, similar to
TT2 of Arabidopsis, which controls PA synthesis in testa. In recessive R-A1 lines, two types of Tamyb10-A1 genes: (1) deletion of the first half of the R2-repeat of the MYB region and (2) insertion of a 2.2-kb transposon belonging
to the hAT family. The Tamyb10-B1 genes of recessive R-B1 lines had 19-bp deletion, which caused a frame shift in the middle part of the open reading frame. With a transient assay
using wheat coleoptiles, we revealed that the Tamyb10 gene in the dominant R-1 allele activated the flavonoid biosynthetic genes. We developed PCR-based markers to detect the dominant/recessive alleles
of R-A1, R-B1, and R-D1. These markers proved to be correlated to known R-1 genotypes of 33 varieties except for a mutant with a single nucleotide substitution. Furthermore, double-haploid (DH) lines
derived from the cross between red- and white-grained lines were found to necessarily carry functional Tamyb10 gene(s). Thus, PCR-based markers for Tamyb10 genes are very useful to detect R-1 alleles. 相似文献
Over the last three decades, successful implementation of the diphtheria vaccination in the developed and developing countries has reduced the infections caused by the toxigenic strains of Corynebacterium diphtheriae, but a concomitant increase in the invasive infections due to the nontoxigenic strains was seen. In addition, the recent reports on the emergence of nontoxigenic toxin gene‐bearing strains, having the potential to revert back to toxigenic form poses a significant threat to human beings. Besides infections caused by C. diphtheriae, the emergence of the respiratory, cutaneous and invasive infections by related pathogenic Corynebacterium species like C. ulcerans and C. pseudotuberculosis, complicate the diagnosis and management of infection. These observations together with the widespread prevalence of diphtheria in the vaccine era, necessitates the strengthening of the epidemiological surveillance and laboratory diagnosis of the pathogen. This review provides the overview of the advantages and limitations of different molecular methods and the role of MALDI‐TOF in the laboratory diagnosis of Diphtheria. The contribution of next generation sequencing technology and different genotyping techniques in understanding the pathogenicity, transmission dynamics and epidemiology of the C. diphtheriae is discussed. 相似文献
Corynebacterium diphtheriae, the causative agent of diphtheria, is well-investigated in respect to toxin production, while little is known about C. diphtheriae factors crucial for colonization of the host. In this study, we investigated strain-specific differences in adhesion, invasion
and intracellular survival and analyzed formation of pili in different isolates. 相似文献
We investigated the relationship between the outer membrane protein OprD2 and carbapenem-resistance in 141 clinical isolates of Pseudomonas aeruginosa collected between January and December 2013 from the First Affiliated Hospital of Anhui Medical University in China. Agar dilution methods were employed to determine the minimum inhibitory concentration of meropenem (MEM) and imipenem (IMP) for P. aeruginosa. The gene encoding OprD2 was amplified from141 P. aeruginosa isolates and analyzed by PCR and DNA sequencing. Differences between the effects of IMPR and IMPS groups on the resistance of the P. aeruginosa were observed by SDS-poly acrylamide gel electrophoresis (SDS-PAGE). Three resistance types were classified in the 141 carbapenem-resistant P. aeruginosa (CRPA) isolates tested, namely IMPRMEMR (66.7%), IMPRMEMS (32.6%), and IMPRMEMS (0.7%). DNA sequencing revealed significant diverse gene mutations in the OprD2-encoding gene in these strains. Thirty-four strains had large fragment deletions in the OprD2gene, in 6 strains the gene contained fragment inserts, and in 96 resistant strains, the gene featured small fragment deletions or multi-site mutations. Only 4 metallo-β-lactamase strains and 1 imipenem-sensitive (meropenem-resistant) strain showed a normal OprD2 gene. Using SDS-PAGE to detect the outer membrane protein in 16 CRPA isolates, it was found that 10 IMPRMEMR strains and 5 IMPRMEMS strains had lost the OprD2 protein, while the IMPSMEMR strain contained a normal 46-kDa protein. In conclusion, mutation or loss of the OprD2-encoding gene caused the loss of OprD2, which further led to carbapenem-resistance of P. aeruginosa. Our findings provide insights into the mechanism of carbapenem resistance in P. aeruginosa. 相似文献
To establish a positive cloning system with a zero background for high-throughput DNA cloning purpose.
Results
The cloning vector, pRI857, and the genomic-library construction vector, pRI857-BAC, were constructed based on the mechanism of expression of the thermo-sensitive cI857 repressor gene that can stringently repress the PR promoter and kanamycin resistance gene (PR-kanR) at 30 °C, but have no effect on PR-kanR gene at 37 °C or at higher temperatures. When the pRI857 vectors were transformed into E. coli with or without a target foreign DNA fragment inserted at the BfrBI site of the cI857 gene, only colonies with the foreign DNA fragment survive. We extended this method to construct a pRI857-BAC vector for genomic library cloning which displays an efficiency of ~107 cfu per µg of genomic DNA, with no empty vectors detected.
Conclusions
Cloning by indirect activation of resistance marker gene represents a novel DNA-capturing system, which can be widely applied for high-throughput DNA cloning.
The Td-based combined vaccine contains only small amounts of the diphtheria toxoid antigen. However, a high level of purity is necessary for this antigen. The diphtheria toxin is produced by growing Corynebacterium diphtheriae in a semisynthetic, casein-based medium in a fermenter. In order to obtain a highly pure diphtheria toxoid, the optimal conditions to express the toxin at 300 Lf/mL in a fermenter culture were determined. When C. diphtheriae was cultivated in a fermenter and a high concentration of toxin was obtained, specific patterns for the pH and dissolved oxygen levels identified. Overall, the fermenter cultivation process was divided into four stages according to variations in the pH. A specific range of KLa in the fermenter (0.0092 ~ 0.0093/sec) was required to produce high level expression of diphtheria toxin. The amount of toxin expression varied significantly according to culture conditions. Agitation and aeration in the fermenter affected toxin expression, even when the optimal KLa value for toxin production was maintained. A previous study has reported that the amounts of agitation and aeration are important factors when cultivating fungus in the fermenter to produce chitinolytic enzyme. A mass production of diphtheria toxoid with a purity level greater than 2,500 Lf/ mgPN was obtained through purification and detoxification from this optimized toxin production. 相似文献
The varitint-waddler mutation A419P renders TRPML3 constitutively active,
resulting in cationic overload, particularly in sustained influx of
Ca2+. TRPML3 is expressed by inner ear sensory hair cells, and we
were intrigued by the fact that hair cells are able to cope with expressing
the TRPML3(A419P) isoform for weeks before they ultimately die. We
hypothesized that the survival of varitint-waddler hair cells is linked to
their ability to deal with Ca2+ loads due to the abundance of
plasma membrane calcium ATPases (PMCAs). Here, we show that PMCA2
significantly reduced [Ca2+]i increase and
apoptosis in HEK293 cells expressing TRPML3(A419P). The deaf-waddler isoform
of PMCA2, operating at 30% efficacy, showed a significantly decreased ability
to rescue the Ca2+ loading of cells expressing TRPML3(A419P). When
we combined mice heterozygous for the varitint-waddler mutant allele with mice
heterozygous for the deaf-waddler mutant allele, we found severe hair bundle
defects as well as increased hair cell loss compared with mice heterozygous
for each mutant allele alone. Furthermore, 3-week-old double mutant mice
lacked auditory brainstem responses, which were present in their respective
littermates containing single mutant alleles. Likewise, heterozygous double
mutant mice exhibited severe circling behavior, which was not observed in mice
heterozygous for TRPML3(A419P) or PMCA2(G283S) alone. Our results provide a
molecular rationale for the delayed hair cell loss in varitint-waddler mice.
They also show that hair cells are able to survive for weeks with sustained
Ca2+ loading, which implies that Ca2+ loading is an
unlikely primary cause of hair cell death in ototoxic stress situations.Varitint-waddler (Va) mice express a mutant isoform (A419P) of the
transient receptor potential channel TRPML3 (murine gene symbol,
Mcoln3) that results in profound hearing loss, vestibular defects
(circling behavior, imbalance, head bobbing, waddling), pigmentation
deficiencies, sterility, and perinatal lethality in homozygous animals
(1). A second Mcoln3
variant (VaJ) that arose in the Va background
carries two mutations (I362T and A419P) and shows a phenotype with reduced
severity, particularly in heterozygous animals
(1). The A419P mutation in
Va and VaJ mice is located in
transmembrane-spanning domain
5(TM5)3 of TRPML3,
where it leads to a constitutively open channel, resulting in highly elevated
[Ca2+]i
(2-5).
In contrast to the effect of the A419P mutation on TRPML3 channel activity,
the single I362T mutation does not appear to affect
[Ca2+]i
(3,
5). When combined with the
A419P mutation, as found in VaJ mice, the constitutive
activity of this mutant TRPML3 isoform is comparable with that of A419P alone
(2-5).Here, we show that HEK293 cells expressing TRPML3-(A419P) or
TRPML3(I362T/A419P) undergo rapid apoptosis. This apoptosis is suppressed by
coexpression of plasma membrane calcium ATPase type 2 (PMCA2). In
varitint-waddler mice, sensory hair cells survive for weeks after birth
(6), which raised the question
of whether this survival could be the result of the hair cells'' ability to
deal with normally transient and localized Ca2+ influx, a feature
that is centered around the high levels of mobile Ca2+ buffers and
PMCA isoforms found in sensory hair cells
(7-10).
We decided to test this hypothesis in vivo by utilizing deaf-waddler
mice that carry a mutation (G283S) in the Atp2b2 gene encoding mutant
PMCA2. Mice homozygous for PMCA2(G283S)
(Atp2b2dfw/dfw) are deaf and have poor
balance (11). Compared with
Atp2b2 knock-out mice, deaf-waddler mice display a milder phenotype
because PMCA2(G283S) retains 30% of its biological activity compared with the
wild-type isoform (12). We
found that sensory hair cell loss, hearing loss, and vestibular dysfunction
were aggravated in mice carrying varitint-waddler and deaf-waddler alleles
compared with animals carrying the single mutant alleles. Our results reveal
that the Ca2+-buffering and Ca2+ extrusion abilities of
hair cells are powerful enough to prevent cell death for weeks, even in the
presence of constitutively active TRPML3(A419P), which is able to induce rapid
apoptosis in other cells. 相似文献
A position-dependent pattern of epidermal cell types is produced during root development in Arabidopsis thaliana. This pattern is reflected in the expression pattern of GLABRA2 (GL2), a homeobox gene that regulates cell differentiation in the root epidermis. GL2 promoter::GUS fusions were used to show that the TTG gene, a regulator of root epidermis development, is necessary for maximal GL2 activity but is not required for the pattern of GL2 expression. Furthermore, GL2-promoter activity is influenced by expression of the myc-like maize R gene (35S::R) in Arabidopsis but is not affected by gl2 mutations. A position-dependent pattern of cell differentiation and GL2-promoter activity was also discovered in the hypocotyl epidermis that was analogous to the pattern in the root. Non-GL2-expressing cell files in the hypocotyl epidermis located outside anticlinal cortical cell walls exhibit reduced cell length and form stomata. Like the root, the hypocotyl GL2 activity was shown to be influenced by ttg and 35S::R but not by gl2. The parallel pattern of cell differentiation in the root and hypocotyl indicates that TTG and GL2 participate in a common position-dependent mechanism to control cell-type patterning throughout the apical-basal axis of the Arabidopsis seedling. 相似文献
Methionine sulfoxide reductases (Msrs) are oxidoreductases that catalyze
thiol-dependent reduction of oxidized methionines. MsrA and MsrB are the best
known Msrs that repair methionine-S-sulfoxide (Met-S-SO) and
methionine-R-sulfoxide (Met-R-SO) residues in proteins,
respectively. In addition, an Escherichia coli enzyme specific for
free Met-R-SO, designated fRMsr, was recently discovered. In this
work, we carried out comparative genomic and experimental analyses to examine
occurrence, evolution, and function of fRMsr. This protein is present in
single copies and two mutually exclusive subtypes in about half of prokaryotes
and unicellular eukaryotes but is missing in higher plants and animals. A
Saccharomyces cerevisiae fRMsr homolog was found to reduce free
Met-R-SO but not free Met-S-SO or dabsyl-Met-R-SO.
fRMsr was responsible for growth of yeast cells on Met-R-SO, and the
double fRMsr/MsrA mutant could not grow on a mixture of methionine sulfoxides.
However, in the presence of methionine, even the triple fRMsr/MsrA/MsrB mutant
was viable. In addition, fRMsr deletion strain showed an increased
sensitivity to oxidative stress and a decreased life span, whereas
overexpression of fRMsr conferred higher resistance to oxidants. Molecular
modeling and cysteine residue targeting by thioredoxin pointed to
Cys101 as catalytic and Cys125 as resolving residues in
yeast fRMsr. These residues as well as a third Cys, resolving
Cys91, clustered in the structure, and each was required for the
catalytic activity of the enzyme. The data show that fRMsr is the main enzyme
responsible for the reduction of free Met-R-SO in S.
cerevisiae.Among the 20 common amino acids in proteins, Met and Cys are the residues
most susceptible to oxidation by reactive oxygen species
(ROS).3 Upon
oxidation, Met forms a diastereomeric mixture of
methionine-S-sulfoxide (Met-S-SO) and
methionine-R-sulfoxide (Met-R-SO). Met-S-SO and
Met-R-SO can be reduced back to Met by MsrA (Met-S-SO
reductase) and MsrB (Met-R-SO reductase), respectively
(1). These enzymes have been
reported to play important roles in the protection of cells and proteins
against oxidative stress
(2–8).
Reversible Met oxidation has also been proposed to scavenge ROS, thereby
protecting cells from oxidative damage
(9–11).
Increased expression of MsrA and MsrB can extend the life span of yeast cells
and fruit flies, whereas deletion of the MsrA gene leads to the reduction in
life span in mice and yeast
(12–14).Previously, three MsrB isozymes and a single MsrA were found in mammals.
MsrB1 (also known as SelR or SelX) is a selenoprotein, which contains
selenocysteine (Sec) in the active site and is localized to cytosol and
nucleus. MsrB2 and MsrB3 are Cys-containing homologs of MsrB1. MsrB2 resides
in mitochondria, whereas human MsrB3 has two alternative splice forms, wherein
MsrB3A localizes to the endoplasmic reticulum and MsrB3B is targeted to
mitochondria (15).The catalytic mechanism of MsrA involves a sulfenic acid intermediate at
the catalytic Cys followed by the formation of a disulfide bond between the
catalytic and resolving Cys. A third Cys may then form a disulfide with the
resolving Cys (16,
17). The resulting disulfide
is reduced by thioredoxin or other oxidoreductases, generating the initial,
reduced form of the protein. X-ray structures of MsrAs from several organisms
have been solved (17,
18).Cys-containing MsrBs (e.g. mammalian MsrB2 and MsrB3) follow the
same mechanism, although the two Msr types have no homology and are
characterized by different structural folds
(19–21).
Sec-containing mammalian MsrB1 has also been characterized and compared with
Cys-containing MsrBs (20).
Interestingly, Cys-containing MsrBs share some active site features
(e.g. conserved residues His77, Val81, and
Asn97, numbering based on mouse MsrB1 sequence), which are absent
in selenoprotein MsrB1s. When these three residues were introduced into the
Sec-containing MsrB1, the enzyme was inactive. However, when the three
residues were introduced into the Cys mutant form of MsrB1, the activity was
partially recovered (20). This
evidence supports the idea that catalytic Cys and Sec require different active
site features.In addition to MsrA and MsrB functions, previous studies suggested the
presence of additional Msr activities in Escherichia coli and yeast
cells, which were especially evident in cells deficient in both enzymes
(14,
21–23).
Recently, Lowther and colleagues
(24) discovered a new enzyme,
designated fRMsr (free Met-R-SO reductase), which catalyzes the
reduction of free Met-R-SO in E. coli. They showed that this
activity is associated with a GAF-like-domain-containing protein. Homologs of
this enzyme were found in other bacteria as well as in eukaryotes, suggesting
that these proteins also could function as fRMsrs. However, none of these
other proteins have been functionally characterized.In this work, we cloned a yeast homolog of bacterial fRMsr and functionally
characterized it with regard to the in vivo function and catalytic
mechanism. In addition, we carried out comparative genomic analyses to examine
evolution of this protein family. The data show that fRMsr is the main enzyme
responsible for the reduction of free Met-R-SO in both prokaryotes
and unicellular eukaryotes. 相似文献
TRPM4 is a non-selective Ca2+-activated cation channel expressed in the heart, particularly in the atria or conduction tissue. Mutations in the Trpm4 gene were recently associated with several human conduction disorders such as Brugada syndrome. TRPM4 channel has also been implicated at the ventricular level, in inotropism or in arrhythmia genesis due to stresses such as ß-adrenergic stimulation, ischemia-reperfusion, and hypoxia re-oxygenation. However, the physiological role of the TRPM4 channel in the healthy heart remains unclear.
Objectives
We aimed to investigate the role of the TRPM4 channel on whole cardiac function with a Trpm4 gene knock-out mouse (Trpm4-/-) model.
Methods and Results
Morpho-functional analysis revealed left ventricular (LV) eccentric hypertrophy in Trpm4-/- mice, with an increase in both wall thickness and chamber size in the adult mouse (aged 32 weeks) when compared to Trpm4+/+ littermate controls. Immunofluorescence on frozen heart cryosections and qPCR analysis showed no fibrosis or cellular hypertrophy. Instead, cardiomyocytes in Trpm4-/- mice were smaller than Trpm4+/+with a higher density. Immunofluorescent labeling for phospho-histone H3, a mitosis marker, showed that the number of mitotic myocytes was increased 3-fold in the Trpm4-/-neonatal stage, suggesting hyperplasia. Adult Trpm4-/- mice presented multilevel conduction blocks, as attested by PR and QRS lengthening in surface ECGs and confirmed by intracardiac exploration. Trpm4-/-mice also exhibited Luciani-Wenckebach atrioventricular blocks, which were reduced following atropine infusion, suggesting paroxysmal parasympathetic overdrive. In addition, Trpm4-/- mice exhibited shorter action potentials in atrial cells. This shortening was unrelated to modifications of the voltage-gated Ca2+ or K+ currents involved in the repolarizing phase.
Conclusions
TRPM4 has pleiotropic roles in the heart, including the regulation of conduction and cellular electrical activity which impact heart development. 相似文献
The earlier established structures of the acidic O-specific polysaccharides from two typical strains of the Shigella dysenteriae bacterium were revised using modern NMR spectroscopy techniques. In particular, the configurations of the glycosidic linkages of GlcNAc (S. dysenteriae type 4) and mannose (S. dysenteriae type 5) residues were corrected. In addition, the location of the sites of non-stoichiometric O-acetylation in S. dysenteriae type 4 was determined: the lateral fucose residue was shown to be occasionally O-acetylated; also, theposition of the O-acetyl group present at the stoichiometric quantity in S. dysenteriae type 5 was corrected. The revised structures of the polysaccharides studied are shown below. The known identity of the O-specific polysaccharide structures of S. dysenteriae type 5 and Escherichia coli O58 was confirmed by 13C NMR spectroscopy and, hence, the structure of the E. coli O58 polysaccharide should be revised in the same manner.